Revealing the Mechanisms and Physiology of Peroxisome Transfer in Stem Cell Therapy

项目摘要（Abstract）细胞间通讯在所有组织和器官中都很重要， 各种机制。细胞因子分泌、间隙连接蛋白、小分子和外泌体已经被研究。 显示促进细胞间细胞质“信息”的交换。另一种新的蜂窝模式 通讯是细胞器转移的通讯，这已被证明发生在线粒体中。过氧体 是小的脂质结合的细胞器，负责代谢的非常长链的脂肪酸， cell.有几种罕见的，但毁灭性的儿童疾病是由于过氧化物酶体缺陷， 肾上腺脑白质营养不良（ALD）。我们有新的证据表明过氧化物酶体可以在细胞类型之间转移。 我们假设过氧化物酶体转移可能是一种机制，使细胞的稳定 含有功能失调的过氧化物酶体本申请的目标是开发一种动物模型，其中 过氧化物酶体在造血细胞内被标记以允许过继转移实验和观察 过氧化物酶体转运在体外和体内。目的1：优化斑马鱼过氧化物酶体转移模型。我们 在斑马鱼造血移植模型中产生的试验数据表明，供体细胞可以转移到 过氧化物酶体到受体造血细胞。如果我们能完全优化它， 在HCT后的细胞剂量和时间方面，我们将确定过氧化物酶体的最佳条件 通过改变细胞剂量和移植后读出的时间，在该模型中发生转移。转让将 通过流式细胞术测定并通过细胞分选和荧光显微镜检查验证。目的2：体外研究 过氧化物酶体在各种细胞类型之间转移的现象，并监测细胞功能的变化。我们 已经建立了多种细胞系，并具有含有过氧化物酶体标签序列的GFP的原代细胞 （PTS-GFP），其允许特异性过氧化物酶体标记。我们将进行共培养实验， 过氧化物酶体转移，并将量化将健康的过氧化物酶体转移到细胞中的生理效应 过氧化物酶体功能失调，测量活性氧和线粒体功能。目标3： 在Rosa基因座的控制下产生表达PTS-GFP的小鼠。这一目标的目标是 PTS-GFP转基因小鼠群体的建立以用作过继免疫中的供体或受体 实验允许我们确定过氧化物酶体转移是否在体内发生。来自供体小鼠的HSPC将 移植到野生型小鼠体内。PTS-GFP小鼠也可以用作受体以确定PTS-GFP小鼠的细胞是否具有免疫原性。 骨髓龛可将过氧化物酶体转移到未标记的供体细胞中。这个项目风险很大， 高回报，因为它的成功将打开一个全新的领域和思维过程到过氧化物酶体，细胞器 传输和蜂窝通信。我们将获得新的知识和建立工具，不仅更好地了解 过氧化物酶体转移，但开发新的策略，药物和小分子，以加强这一进程， 可能会影响我们如何治疗像ALD这样的破坏性过氧化物酶体疾病。

Project Summary (Abstract). Cell-to-cell communication is important in all tissues and organs and occurs by a variety of mechanisms. Cytokine secretion, gap junction proteins, small molecules, and exosomes have been shown to facilitate the exchange of cytoplasmic “information” between cells. Another novel mode of cellular communication is that of organelle transfer which has been shown to occur with mitochondria. Peroxisomes are small lipid bound organelles which are responsible for the metabolism of very long chain fatty acids in the cell. There are several rare, but devastating childhood diseases that are a result of peroxisomal defects such adrenoleukodystrophy (ALD). We have new evidence that peroxisomes can be transferred between cell types. We hypothesize that peroxisomal transfer could be a mechanism that allows for the stabilization of cells harboring dysfunctional peroxisomes. The goal of this application is to develop an animal model in which peroxisomes are labeled within hematopoietic cells to allow adoptive transfer experiments and observation of peroxisomal transport in vitro and in vivo. Aim 1: Optimize the zebrafish model of peroxisomal transfer. Our pilot data, generated in a zebrafish model of hematopoietic transplant, indicate that donor cells can transfer peroxisomes to recipient hematopoietic cells. It would be ideal to understand if we can fully optimize this system in terms of cell dose and time after HCT. We will determine the optimal conditions for peroxisome transfer to occur in this model by varying the cell doses and time after transplant readout. Transfer will be determined by flow cytometry and verified by cell sorting and fluorescent microscopy. Aim 2: Study the in vitro phenomenon of peroxisomal transfer between various cell types and monitor changes in cellular function. We have created a variety of cell lines and have primary cells with GFP containing a peroxisome tag sequence (PTS-GFP) which allows for specific peroxisome labeling. We will perform co-culture experiments to observe peroxisomal transfer and will quantify the physiological effects of transferring healthy peroxisomes into cells with dysfunctional peroxisomes measuring reactive oxygen species and mitochondrial function. Aim 3: Generation of a PTS-GFP expressing mouse under the control of the Rosa locus. The goal of this aim is the establishment of a colony of PTS-GFP transgenic mice to be used as donors or recipients in adoptive experiments allowing us to determine if peroxisomal transfer occurs in vivo. HSPC from donor mice will transplanted into wild-type mice. PTS-GFP mice can also be used as recipients to determine if cells of the marrow niche can transfer peroxisomes into unlabeled donor cells. This project carries both high risk and high reward, as its success will open an entirely new field and thought process into peroxisomes, organelle transfer, and cellular communication. We will gain new knowledge and build tools to not only better understand peroxisomal transfer, but develop new strategies, drugs, and small molecules to enhance the process which could impact how we treated devastating peroxisomal disease like ALD.